Discovery of a conserved pathway for endocytic internalization in a simple organism amenable to powerful genetic, genomic, biochemical and cell biological analysis created unprecedented opportunities to develop a comprehensive understanding of this complex process, and to reveal underlying molecular mechanisms. An integrated approach, combining image analysis, functional genomics, proteomics, biochemistry and theoretical modeling, will elucidate the molecular mechanisms that underlie the endocytic pathway. These studies will increase understanding of actin's role in endocytosis, and how the order and timing of events in the endocytic pathway are achieved. Understanding how dynamic actin assembly is coupled to plasma membrane- associated processes is critical for understanding human health threats including metastasis, loss of proliferation regulation, and pathogen entry into cells. Focused tests of specific hypotheses, and global investigations to reveal holistic operating principles for the pathway, will be balanced. The dynamics of at least 8 newly identified endocytic proteins will be mapped onto the previously described endocytic pathway, and quantitative imaging of endocytosis in mutants of these proteins will identify their biological functions. A screen for mutants with altered sensitivity to the endocytosed yeast killer toxin, K28, coupled with real-time analysis of K28 endocytosis, will identify new endocytic proteins and intracellular sorting mechanisms. Imaging-based analysis of endocytic protein dynamics in mutants of endocytic protein ubiquitination will reveal mechanisms that coordinate events in the pathway. Studies on the functions of the myosin/WASP and scission endocytic protein modules, rather than on individual proteins, will make approachable the otherwise daunting task of deciphering the functions of over 60 endocytic proteins. A combined biochemical, genetic and image-based approach will identify the mechanisms and protein-protein and protein-lipid interactions that recruit these protein modules to the endocytic sites, and that regulate their activities. The ultrastructure of the endocytic machinery at different stages of the endocytic pathway will be determined using cryo electron microscopy on wild-type and mutant cells. The membrane geometry and the ultrastructure of the protein coat and its associated actin filaments, will be determined. Immuno-EM will reveal how different proteins are organized at the endocytic site. Theoretical modeling combined with experimental measurements of protein function, dynamics and ultrastructural organization, will develop the first coherent and quantitative model of endocytic dynamics that can recapitulate all of the key events. Quantitative design principles from the resulting model will likely apply to other cytoskeletal and membrane trafficking processes. Hypotheses generated by theoretical modeling will be tested experimentally. ) PUBLIC HEALTH RELEVANCE: Understanding how dynamic actin assembly is coupled to plasma membrane-associated processes is critical for understanding human health threats including metastasis, loss of proliferation regulation, and pathogen entry into cells. Studies on toxin entry into yeast cells and toxin-mediated cell killing may suggest novel therapeutic strategies for treatment of bacterial infections.)